Isolated FrpB nucleic acid molecule and vaccine

ABSTRACT

The present invention provides an isolated nucleic acid molecule that encodes an amino acid sequence comprising a FrpB protein. The invention also provides vaccine compositions capable of protecting a mammal against infection by  N. gonorrhoeae  or  N. meningitidis  comprising the FrpB protein encoded by the isolated nucleic acid of the invention and a pharmaceutically acceptable carrier.

[0001] This specification is a continuation-in-part of Ser. No.08/418,964 filed Apr. 7, 1995, which is incorporated herein byreference.

[0002] This invention was made in the course of work supported by PublicHealth Service Grant U01 Al 31496 and the Genetics Curriculum traininggrant 5 T32 GM07092 from the National Institutes of Health. Proteinsequencing performed at the UCLA Protein Microsequencing Facility wasaided by a BRS Shared Instrumentation Grant (I S10RR05554-01) from theNational Institutes of Health. The United States government has certainrights in this invention.

BACKGROUND OF THE INVENTION

[0003] FrpB has been described as a 70 kD major iron-regulated,outer-membrane protein common to N. gonorrhoeae and N. menigitidis (16,21). The iron uptake systems of N. meningitidis and N. gonorrhoeae aresimilar (3,17).

[0004] Previous studies showed that FrpB is surface exposed andimmunogenic in vivo (1,16, 41). Polyclonal and some monoclonal anti-FrpBantibodies recognize the denatured protein on Western blots of nearlyall gonococcal and meningococcal isolates tested (16 and thisinvention). Other monoclonal antibodies directed against meningococcalFrpB are bactericidal and strain specific (41). Nevertheless, the sizeof FrpB appears to be well conserved.

[0005] FrpB is useful as a vaccine because of its surface exposure(1,16,41), partial antigenic conservation (8,16), and susceptibility toattack by bactericidal antibodies (41). The cloning and sequencing ofthe frpB gene of this invention has made possible the production of avaccine against infection in mammals by N. gonorrhoeae or N.meningitidis.

SUMMARY OF THE INVENTION

[0006] The present invention provides an isolated nucleic acid moleculethat encodes an amino acid sequence comprising a FrpB protein.

[0007] The invention also provides a method of producing a vaccinecomposition that protects a mammal from infection by N. gonorrhoeae orN. meningitidis comprising combining the FrpB protein encoded by theisolated nucleic acid of the invention with a pharmaceuticallyacceptable carrier.

[0008] The invention further provides a vaccine composition capable ofprotecting a mammal against infection by N. gonorrhoeae or N.meningitidis, the vaccine composition comprising the FrpB proteinencoded by the isolated nucleic acid of the invention and apharmaceutically acceptable carrier.

[0009] In addition, the invention provides antibodies directed to anepitope of the FrpB protein encoded by the isolated nucleic acidsequence of the invention.

[0010] The invention also provides a method of detecting an antibodyspecific for N. gonorrhoeae or N. meningitidis in a sample comprisingcontacting the sample with a FrpB protein encoded by the isolatednucleic acid sequence of the invention under conditions to form acomplex between the polypeptide and the antibody; and detecting anycomplex so formed.

[0011] Furthermore, the invention provides a method of treating a mammalinfected by N. gonorrhoeae or N. meningitidis comprising administeringto the mammal an antibody of the invention, wherein the antibody isdirected to an epitope of an N. gonorrhoeae or N. meningitidis FrpBprotein.

BRIEF DESCRIPTION OF THE FIGURES

[0012]FIG. 1 Oligonucleotide MB.3 is shown 3′ to 5′ and corresponds tonon-coding strand. The frpB sequence presented in this figure isdeposited with GenBank under the accession number U13980.

[0013]FIG. 2 Restriction map of frpB clones. The position of the frpBORF is indicated below the physical map by the stippled box. Onlyrelevant cloning sites are shown C, Cla I; D, Dra I; E, EcoR I; M, MluI. Also shown is the position of oligonucleotide MB.3, which was deducedfrom the amino-terminal amino acid sequence of the mature protein.

[0014]FIG. 3 Nucleotide sequence of the gonococcal frpB gene from strainFA19. Single letter codes for deduced amino acid sequence are shownbelow the nucleotide sequence. Asterisk indicates termination codon.Solid bar below nucleotide sequence indicates putative Fur box. Putative−10 and −35 sequences are boxed. RBS indicates ribosome binding site.Solid triangle shows Bgl I site of Ω insertion. Vertical arrow indicatessignal peptidase I cleavage site. Inverted horizontal arrows indicateinverted repeat.

[0015]FIG. 4 Southern-blot analysis of FA19 and FA6807 DNA. Panel A wasprobed with pUNCH319-specific fragment. Panel B was probed with the Ωfragment. Lanes 1 contain FA19 DNA digested with HincII and lanes 2contain FA6807 DNA digested with Hincil. Ω fragment is 2 kb. Molecularweight markers are shown in kilobases (kB).

[0016]FIG. 5 Western blot of FA19 and FA6807 membranes. Blot was probedwith anti-FrpB monoclonal antibody, W.6. Lanes 1 and 2 are FA19; lanes 3and 4 are FA6807. Lanes 1 and 3 contain total membranes prepared fromiron-sufficient cultures; lanes 2 and 4 contain total membranes fromiron-deficient cultures. Approximate locations of molecular massstandards are indicated at left in kilodaltons.

[0017]FIG. 6 Growth of FA19 and FA6807 in CDM in the presence ofvariable concentrations of aerobactin. Graph A represents FA19; graph Brepresents FA6807. (filled-in Δ), 100 uM citrate; (▪), 2.5 uM Tf; (Δ), 3uM aerobactin; (), 1 uM aerobactin; (□), 0.3 uM aerobactin; and (◯), noiron source.

[0018]FIG. 7 ⁵⁵Fe uptake from ⁵⁵Fe-heme and ⁵⁵Fe-Tf. Solid columnsrepresent mean uptake from heme and open columns represent mean uptakefrom Tf. 100% uptake determined from average FA19 uptake experiment.Standard deviations are indicated by error bars. Genotypes are FA19 wildtype, FA6807 (frpB), and FA6747 (tpbA).

[0019]FIG. 8 Reconstruction of frpB in pACYC184. Relevant sites are B,BamH I; C, Cla I; D, Dra I; M, Mlu I; and X, Xba I. Solid arrowrepresents chloramphenicol acetyl transferase (Cm), stripped arrowrepresents tetracycline resistance gene (Tc), solid bar representspACYC184 origin of replication (Ori), stippled boxes represent frpBcoding sequences, stippled arrow indicates entire frpB coding regions,open boxes represent DNA 5′ and 3′ of frpB. frpB′ and frpB″ representpartial frpB coding sequences.

[0020]FIG. 9 Growth of RK1065 (pACYC184) and RK1065 (pUNCH331) on hemeplates. Plate 1 contains heme only. Plate 2 contains heme andd-aminolevulinic acid. A is RK1065 (pACYC184) and B is RK1065(pUNCH331). Antibiotic discs are E., erythromyocin; N, novobiocin; andR, rifampicin.

[0021]FIG. 10 Nucleotide sequence of the gonococcal frpB gene fromstrain FA1090. The three letter codes for deduced amino acid sequenceare shown below the nucleotide sequence. Three asterisks indicatetermination codon.

DETAILED DESCRIPTION OF THE INVENTION

[0022] The subject invention provides an isolated nucleic acid moleculethat encodes an amino acid sequence comprising at least a portion of aFrpB protein. In one embodiment of this invention, the isolated nucleicacid molecule is DNA. In other embodiments of this invention, theisolated nucleic acid molecule is cDNA or RNA. In a preferred embodimentof this invention, the isolated nucleic acid molecule comprises asequence that is the same as or substantially the same as at least aportion of the nucleotide sequence shown in FIG. 3. In a more preferredembodiment, the isolated nucleic acid molecule comprises a sequence thatis the same as the nucleotide sequence shown in FIG. 3.

[0023] The invention also provides a FrpB protein comprising the aminoacid sequence encoded by the isolated nucleic acid molecules describedabove. Preferably, the amino acid sequence encodes an antigenic, andmore preferably, an immunogenic FrpB. As used herein, antigenic meansthat the FrpB induces specific antibodies in a mammal, and immunogenicmeans that the FrpB induces an immune response in a mammal.

[0024] As used herein, the term “FrpB” means Fe-regulated protein B andencompasses any polypeptide having an amino acid sequence identical, orsubstantially identical, to the amino acid sequence of anaturally-occurring FrpB, as well as antigenic fragments thereof. TheFrpB nucleic acid and amino acid sequences in the various strains of N.gonorrhoeae and N. meningitidis are homologous, but exhibit slightdifferences in their sequences, for example, the nucleic acid and aminoacid differences between the homologous strains FA19 and FA1090 shown inFIG. 3 and FIG. 10, respectively.

[0025] In addition, FrpB encompasses equivalent antigenic polypeptideswhose amino acid sequence varies from a naturally-occurring FrpB by oneor more amino acid, either internally such as a point mutation, or byaddition or deletion at the COOH⁻ terminus or NH₂ terminus or both. Anamino acid sequence that is substantially the same as another sequence,but that differs from the other sequence by one or more substitutions,additions and/or deletions, is considered to be an equivalent sequence.Preferably, less than 25%, more preferably less than 10%, and mostpreferably less than 5% of the number of amino acid residues in asequence are substituted for, added to, or deleted from the proteins ofthe invention.

[0026] For example, it is known to substitute amino acids in a sequencewith equivalent amino acids. Groups of amino acids generally consideredto be equivalent are:

[0027] (a) Ala(A) Ser(S) Thr(T) Pro(P) Gly(G);

[0028] (b) Asn(N) Asp(D) Glu(E) Gln(Q);

[0029] (c) His(H) Arg(R) Lys(K);

[0030] (d) Met(M) Leu(L) IIe(I) Val(V); and

[0031] (e) Phe(F) Tyr(Y) Trp(W).

[0032] Such FrpB equivalents include analogs that induce an immuneresponse in a mammal comparable to that of natural FrpB. In addition,such equivalents are immunologically cross-reactive with theircorresponding FrpB protein.

[0033] A FrpB protein fragment preferably contains sufficient amino acidresidues to define an epitope of the antigen. The fragment may, forexample, be a minigene encoding only the epitope. Methods for isolatingand identifying immunogenic fragments from known immunogenic proteinsare described by Salfeld et al. (72) and by Isola et al. (73).

[0034] If the fragment defines a suitable epitope, but is too short tobe immunogenic, it may be conjugated to a carrier molecule. Somesuitable carrier molecules include keyhole limpet hemocyanin, Igsequences, TrpE, and human or bovine serum albumen. Conjugation may becarried out by methods known in the art. One such method is to combine acysteine residue of the fragment with a cysteine residue on the carriermolecule.

[0035] In a preferred embodiment, FrpB of FA19 is or is an equivalent ofthe approximately 73 kD outer membrane FrpB protein that is part of theiron regulon of Neisseria gonorrhoeae or of Neisseria meningitidis.Determinations whether two amino acid sequences are substantiallyhomologous may be based on FASTA searches in accordance with Pearson andLipman (74).

[0036] The FrpB of the present invention may be prepared by methodsknown in the art. Such methods include, for example, (a) isolating FrpBdirectly from Neisseria gonorrhoeae or Neisseria meningitidis; and (b)using the nucleic acid molecule of the invention encoding FrpB toproduce recombinant FrpB.

[0037] (a) Direct Isolation of FrpB:

[0038] The FrpB may be isolated directly from Neisseria gonorrhoeae orNeisseria meningitidis by methods known in the art. First, gonococcal ormeningococcal outer membranes are isolated and prepared by knownmethods. The methods described by West and Sparling (75) and bySchryvers and Morris (76) are suitable.

[0039] The isolated membrane FrpB proteins or fragments may besolubilized by known methods, such as the addition of detergents.Commonly used detergents include Octyl-B-Glucoside, Chaps, Zwittergent3.14 or Triton-X. The use of detergents to enhance solubility ofmembrane proteins is described by Jones et al. (77), Helenius et al.(78), and Hjelmeland and Chrambach (79).

[0040] The FrpB proteins or fragments are isolated from the solubilizedmembrane fraction by standard methods. Some suitable methods includeprecipitation and liquid chromatographic protocols such as ion exchange,hydrophobic interaction and gel filtration. See, for example, MethodsEnzymol. (80) and Scopes (81).

[0041] Purified material may also be obtained by separating the proteinor fragment on preparative SDS-PAGE gels, slicing out the band ofinterest and electroeluting the protein from the polyacrylamide matrixby methods known in the art. The detergent SDS is removed from theprotein by known methods, such as by dialysis or the use of a suitablecolumn, such as the Extracti-Gel column from Pierce.

[0042] (b) Using Nucleic Acid Molecule of the Invention to Produce FrpB:

[0043] Alternatively, recombinant methods known in the art may be usedfor preparing FrpB. For example, FrpB may be produced from the isolatedor synthesized nucleic acid molecule of the invention that encodes atleast a portion of FrpB; cloning the DNA in a suitable host; expressingthe DNA in the host; and harvesting FrpB. (See Sambrook et al. (82)).

[0044] Using standard methods of nucleic acid isolation, DNA can beobtained from strains that have been deposited with the American TypeCulture Collection, Rockville, Md. FA1090 (ATCC Accession No.) wasdeposited on Apr. 8, 1996, in accordance with the Budapest Treaty.Strain FA19 (ATCC Accession No. 55073) was deposited earlier on Jul. 12,1996, also in accordance with the Budapest Treaty.

[0045] The DNA may also be synthesized chemically from the fournucleotides in whole or in part by methods known in the art. Suchmethods include those described by Caruthers in Science 230, 281-285(1985).

[0046] If necessary a full length DNA may also be produced by preparingoverlapping double-stranded oligonucleotides, filling in the gaps, andligating the ends together. The DNA may be cloned in a suitable hostcell and expressed. The DNA and protein may be recovered from the hostcell. See, generally, Sambrook et al, “Molecular Cloning,” SecondEdition, Cold Spring Harbor Laboratory Press (1987).

[0047] The invention provides a vector which comprises the nucleic acidmolecule described above which encodes an amino acid sequence comprisingat least a portion of FrpB. Suitable vectors comprise, but are notlimited to, a plasmid or a virus. This vector may be transfected into asuitable host cell to form a host vector system for the production ofFrpB or of a polypeptide having the biological activity of at least aportion of a FrpB antigenic polypeptide.

[0048] Cloning vectors may comprise segments of chromosomal,non-chromosomal and synthetic DNA sequences. Some suitable prokaryoticvectors include plasmids from E. coli, such as colE1, pCR1, pBR322,pMB9, and RP4. Prokaryotic vectors also include derivatives of phage DNAsuch as M13, f1, and other filamentous single-stranded DNA phages.

[0049] Vectors for expressing proteins in bacteria, especially E. coli,are also known. Such vectors include pK233 (or any of the tac family ofplasmids), T7, and lambda P_(L). Examples of vectors that express fusionproteins include the PATH vectors described by Dieckmann and Tzagoloff(83). These vectors contain DNA sequences that encode anthranilatesynthetase (TrpE) followed by a polylinker at the carboxy terminus.Other expression vector systems are based on beta-galactosidase (pEX);maltose binding protein (pMAL); and glutathione S-transferase (PGST)—seeGene (84) and Peptide Research (85).

[0050] Vectors useful in yeast are available. A suitable example is the2μ plasmid.

[0051] Suitable vectors for use in mammalian cells are also known. Suchvectors include well-known derivatives of SV-40, adenovirus,retrovirus-derived DNA sequences and vectors derived from combination ofplasmids and phage DNA.

[0052] Further eukaryotic expression vectors are known in the art (e.g.,P. J. Southern and P. Berg (86); S. Subramani et al (87); R. J. Kaufmannand P. A. Sharp (88); S.I. Scahill et al (89); G. Urlaub and L. A.Chasin (90).

[0053] The expression vectors preferably contain at least one expressioncontrol sequence that is operatively linked to the DNA sequence orfragment to be expressed. The control sequence is inserted in the vectorin order to control and to regulate the expression of the cloned DNAsequence. Examples of useful expression control sequences are the lacsystem, the trp system, the tac system, the trc system, major operatorand promoter regions of phage lambda, the control region of f1 coatprotein, the glycolytic promoters of yeast, e.g., the promoter for3-phosphoglycerate kinase, the promoters of yeast acid phosphatase,e.g., Pho5, the promoters of the yeast alpha-mating factors, andpromoters derived from polyoma, adenovirus, retrovirus, and simianvirus, e.g., the early and late promoters or SV40, and other sequencesknown to control the expression of genes of prokaryotic or eukaryoticcells and their viruses or combinations thereof.

[0054] Suitable expression hosts include well-known prokaryotic andeukaryotic cells. Some suitable prokaryotic hosts include, for example,E. coli, such as E. coli SG-936, E. coli HB 101, E. coli W3110, E. coliX1776, E. coli X2282, E. coli DHI, and E. coli MRCI, Pseudomonas,Bacillus, such as Bacillus subtilis, and Streptomyces. Suitableeukaryotic cells include yeasts and other fungi, insect, animal cells,such as COS cells and CHO cells, human cells and plant cells in tissueculture.

Vaccines

[0055] FrpB encoded by a nucleic acid molecule of this invention hasparticular utility as a vaccine that protects a mammal from infection byN. gonorrhoeae or N. meningitidis, since the FrpB unexpectedly inducesan effective immune response when presented to the immune system thatprotects from or prevents infection by N. gonorrhoeae or N.meningitidis. To protect from infection by N. gonorrhoeae, the FrpB ispreferably substantially the same, as defined above, as at least aportion of the FrpB of N. gonorrhoeae. To protect from infection by N.meningitidis, the FrpB is preferably substantially the same, as definedabove, as at least a portion of the FrpB of N. meningitidis. The immuneresponse may also produce a therapeutic effect in an already infectedmammal. The mammal is preferably a human.

[0056] The invention provides a vaccine composition which comprises theFrpB protein encoded by a nucleic acid of the invention and apharmaceutically acceptable carrier, such as saline, sterile water,phosphate buffered saline solution, liposomes and emulsions. Otherbuffering and dispersing agents and inert non-toxic substances suitablefor delivery to a mammal may be incorporated in the vaccine compositionand are well known to those skilled in the art. The compositions may besterilized by conventional sterilization techniques.

[0057] Adjuvants, which facilitate stimulation of the host's immuneresponse, may be used in the vaccine compositions. Such adjuvants mayinclude, for example, muramyl peptides, lymphokines, such as interferon,interleukin-1 and interleukin-6, or bacterial adjuvants. The adjuvantmay comprise suitable particles onto which the mutant or wild-type FrpBprotein is adsorbed, such as aluminum oxide particles. These vaccinecompositions containing adjuvants may be prepared as is known in theart.

[0058] The concentration of FrpB in the composition may vary dependingon, for example, fluid volume or antigenicity, and in accordance withthe particular mode of administraton chosen. The invention furtherprovides a method of protecting a mammal against infection by N.gonorrhoeae or N. meningitidis comprising administering to the mammalthe vaccine composition of the invention. The vaccine may beadministered to a mammal by methods known in the art. Such methodsinclude, for example, oral, intravenous, intraperitoneal, subcutaneous,intramuscular, topical, or intradermal administration.

[0059] This invention also provides a method of producing the abovevaccine composition by combining FrpB with a pharmaceutically acceptablecarrier, and preferably, also with an adjuvant, as defined above.

FrpB Antibodies

[0060] The invention provides antibodies raised against FrpB epitopesencoded by at least a portion of the isolated nucleic acid sequence ofthe invention. The antibodies are preferably monoclonal. Monoclonalantibodies may be produced by methods known in the art. These methodsinclude the immunological method described by Kohler and Milstein (91)and the recombinant DNA method described by Huse et al. (92).

[0061] Mammals infected with N. gonorrhoeae or N. meningitidis may betreated by administering an antibody of the invention. Preferably, anantibody raised against a polypeptide comprising an amino acid sequencepresent in N. gonorrhoeae or N. meningitidis is preferred.

[0062] For therapeutic purposes, the antibodies are preferablyneutralizing antibodies that significantly inhibit the growth of or killthe bacterial cells in vitro or in vivo. Growth of the bacteria issignificantly inhibited in vivo if the inhibition or neutralization issufficient to prevent or reduce the symptoms of the disease of a mammalinfected with the disease.

[0063] Neutralizing antibodies may also be used to produceanti-idiotypic antibodies useful as vaccines for immunizing mammalsinfected with N. gonorrhoeae or N. meningitidis. Anti-idiotypicantibodies are prepared in accordance with methods known in the art.

Detecting FrpB Using Probes

[0064] The invention also provides a method of detecting FrpB in asample using a probe specific for a FrpB polypeptide. The probe may bean antibody described above. Methods are known for detectingpolypeptides with antibodies. For example, a polypeptide may beimmobilized on a solid support. Immobilization of the polypeptide mayoccur through an immobilized first antibody specific for thepolypeptide. The immobilized first antibody is incubated with a samplesuspected of containing the polypeptide. If present, the polypeptidebinds to the first antibody.

[0065] A second antibody, also specific for the polypeptide, binds tothe immobilized polypeptide. The second antibody may be labeled bymethods known in the art. Non-immobilized materials are washed away, andthe presence of immobilized label indicates the presence of thepolypeptide. This and other immunoassays are described by David, et al.,in U.S. Pat. No. 4,376,110 assigned to Hybritech, Inc., La Jolla, Calif.

[0066] The probe may also be a nucleic acid molecule that recognizes aFrpB nucleic acid molecule of the invention. Methods for determiningwhether a nucleic acid molecule probe recognizes a specific nucleic acidmolecule in a sample are known in the art. Generally, a labeled probethat is complementary to a nucleic acid sequence suspected of being in asample is prepared. The presence of probe hybridized to the targetnucleic acid molecule indicates the presence of the nucleic acidmolecule. Suitable methods are described by Schneider et al in U.S. Pat.No. 4,882,269, which is assigned to Princeton University, and by Segevin PCT Application WO 90/01069, which is assigned to ImClone SystemsIncorporated.

[0067] The probes described above are labeled in accordance with methodsknown in the art. Methods for labeling antibodies have been described,for example, by Hunter and Greenwood (93) and by David et al. (94).Additional methods for labeling antibodies have been described in U.S.Pat. Nos. 3,940,475 and 3,645,090. Methods for labeling oligonucleotideprobes have been described, for example, by Leary et al (95); Renz andKurz (96); Richardson and Gumport (97); Smith et al. (98); and Meinkothand Wahl (99).

[0068] The label may be radioactive. Some examples of useful radioactivelabels include ³²P, 125 I, 131I, and ³H. Use of radioactive labels havebeen described in U.K. 2,034,323, U.S. Pat. No. 4,358,535, and U.S. Pat.No. 4,302,204.

[0069] Some examples of non-radioactive labels include enzymes,chromophors, atoms and molecules detectable by electron microscopy, andmetal ions detectable by their magnetic properties.

[0070] Some useful enzymatic labels include enzymes that cause adetectable change in a substrate. Some useful enzymes and theirsubstrates include, for example, horseradish peroxidase (pyrogallol ando-phenylenediamine), beta-galactosidase (fluoresceinbeta-D-galactopyranoside), and alkaline phosphatase(5-bromo-4-chloro-3-indolyl phosphate/nitro blue tetrazolium). The useof enzymatic labels have been described in U.K. 2,019,404, EP 63,879,and by Rotman (100).

[0071] Useful chromophores include, for example, fluorescent,chemiluminescent, and bioluminescent molecules, as well as dyes. Somespecific chromophores useful in the present invention include, forexample, fluorescein, rhodamine, Texas red, phycoerythrin,umbelliferone, and luminol.

[0072] The labels may be conjugated to the antibody or nucleotide probeby methods that are well known in the art. The labels may be directlyattached through a functional group on the probe. The probe eithercontains or can be caused to contain such a functional group. Someexamples of suitable functional groups include, for example, amino,carboxyl, sulfhydryl, maleimide, isocyanate, isothiocyanate.

[0073] The label may also be conjugated to the probe by means of aligand attached to the probe by a method described above and a receptorfor that ligand attached to the label. Any of the known ligand-receptorcombinations is suitable. The biotin-avidin combination is preferred.

[0074] The polypeptide of the invention may be used to detect thepresence of antibodies specific for N. gonorrhoeae or N. meningitidis ina sample. The method comprises preparing a polypeptide containing asegment having an amino acid sequence that is substantially the same asa FrpB from either N. gonorrhoeae to detect antibodies to N. gonorrhoeaeor N. meningitidis to detect antibodies to N. meningitidis. Thepolypeptide may be prepared as described above.

[0075] The sample may, for example, be from a patient suspected of beinginfected with N. gonorrhoeae or N. meningitidis. Suitable assays areknown in the art, such as the standard ELISA protocol described by R. H.Kenneth (101).

[0076] Briefly, plates are coated with antigenic polypeptide at aconcentration sufficient to bind detectable amounts of the antibody.After incubating the plates with the polypeptide, the plates are blockedwith a suitable blocking agent, such as, for example, 10% normal goatserum. The sample, such as patient sera, is added and titered todetermine the endpoint. Positive and negative controls are addedsimultaneously to quantitate the amount of relevant antibody present inthe unknown samples. Following incubation, the samples are probed withgoat anti-human Ig conjugated to a suitable enzyme. The presence ofanti-polypeptide antibodies in the sample is indicated by the presenceof the enzyme.

[0077] The following Examples section is set forth to aid in anunderstanding of the invention. This section is not intended to, andshould not be construed to, limit in any way the invention as set forthin the claims which follow thereafter.

EXAMPLES

[0078] Strains and growth conditions. Bacterial strains used in thisexperiment are described in Table 1. Neisseria strains were routinelycultured on GCB media (Difco Laboratories) containing Kellogg'ssupplements 1 and 11 (29) and grown overnight at 35° C. in an atmosphereof 5%CO₂. Antibiotic selection employed chloramphenicol at 1 μg/ml formTn3(Cm)(51) mutagenized strains and streptomycin at 100 μg/ml for Ω(44) mutagenized strains.

[0079] For western blot analysis of total membrane proteins ofiron-stressed gonococci, cells were grown in CDM as previously described(13). Cultures were made iron replete as indicated by the addition of100 uM ferric citrate.

[0080]E. coli strains were routinely cultured on Luria-Bertani (LB)media (47). Antibiotic selection was 100 μg/ml ampicillin, 100 μg/mlstreptomycin, 40 μg/ml kanamycin, and/or 30 μg/ml choiramphenicol.8-aminolevulinic acid was used at 30 μg/ml and heme at 50 μg/ml. E. colicultures were iron stressed by the addition of 200 μM 2,2-diyridyl(Sigma Chemical Co., St. Louis, Mo.). Deferoxamine mesylate (desferal)was obtained from Ciba-Geigy (Basel, Switzerland).

[0081] SDS-PAGE and Western Blotting. SDS-PAGE was performed in 7.5%polyacrylamide resolving gel and 4.5% polyacrylamide stacking gel.Electrophoresis was carried out at either 40 mA for one gel, or 80 mAfor two gels in the discontinuous buffer system of Laemmli (32).Transfer and development were as described previously (23,61).

[0082] Preparation of polyclonal antisera and monoclonal antibodies.Preparation of polyclonal antisera was described previously (8).Anti-FrpB monoclonal antibodies were generated by methods describedpreviously (60).

[0083] DNA isolation, digestion, and Southern blot analysis. ChromosomalDNA was purified by CsC1-gradient centrifugation according to themethods of Stern et al. (54). Plasmids were purified by either CsC1centrifugation or according to the instructions provided in the MagicMiniprep™ DNA Purification Kit (Promega; Madison Wis.). Southernblotting and DNA hybridizations were performed as previously described(13). Restriction enzymes, Kienow fragment of DNA polymerase I, and T4DNA ligase were purchased from New England Biolabs (Beverly, Mass.) orBethesda Research Laboratories (Gaithersburg, Md.) and were usedaccording to the manufacturer's specifications. λ-ZapII and pBluescriptII SK+ were obtained from Stratagene (La Jolla, Calif.).

[0084] DNA sequencing and sequence analysis. CsCI-purified pUNCH319 andpUNCH325 were used as templates for double-stranded DNA sequencing (31)using United States Biochemical Sequenase and the dideoxy chaintermination procedure of Sanger et al. (48). Both dG- and dl-labelingreactions were carried out for all primers. Both strands of pUNCH319were sequenced using vector-specific or insert-specific primers.Exonuclease III/Exo VII nested deletions (40) were generated from theMlu end of pUNCH325 and vector-specific primers were used to sequenceindividual deletion clones. Internal primers were used to sequence gapsbetween clones as well as the opposite strand. DNA sequences wereanalyzed with the Genetics Computer Group software package (15)(University of Wisconsin).

[0085] Mutagenesis and gonococcal transformation. pHP45 Ω (44) was usedto insertionally inactivate frpB. pUNCH321 was digested with Bgl I andends were repaired with Klenow. pHP45 Ω was digested with Sma I and the2.0 kb Ω fragment was isolated from an agarose gel according to theinstructions provided in the Geneclean II® Kit (Bio101 Inc. La Jolla,Calif.). Transformation of plasmid DNA into FA19 was as previouslydescribed (7).

[0086] Preparation of FrpB for amino-terminal sequence analysis.N-lauroylsarcosine (Sigma) insoluble membrane fractions were preparedfrom iron-stressed gonococcal strain UU1008 and protein concentrationwas determined by a bicinchoninic acid assay (BCA) (Pierce, Rockford,Ill.). Two hundred micrograms of protein was loaded into a preparativewell of a 7.5% SDS-polyacryamide gel, poured 24 hours previously topermit TEMED (N,N,N′,N′-tetramethylethylenediamine) and APS (ammoniumpersulfate) to evaporate. Electrophoresis was carried out at 40 mAconstant current using the discontinuous buffer system of Laemmli (32).The gel was soaked for 15 minutes in transfer buffer (13) beforetransferring. PVDF (polyvinylidene difluoride) membrane was placed in100% methanol for two seconds, transferred to distilled deionized water(ddH₂O) for five minutes, and soaked in transfer buffer for 10 minutesprior to transfer. Transfer was for three and a half hours at 90 mA in asubmerged trans-blot apparatus (BioRad, Richmond, Calif.). Subsequent totransfer, the PVDF membrane was stained for five minutes in 0.1%Coomassie Brilliant Blue, 20% methanol, and 10% acetic acid to visualizeproteins and destained for 10 minutes in ddH2O with one change. Filterwas frozen at −20° C. overnight. FrpB was identified by molecular weightand the amino-terminal amino acid sequence of the protein on the filterwas determined by the Protein Microsequencing Facility at UCLA.

[0087]⁵⁵Fe uptake assays. Data were compiled from three individualexperiments performed in triplicate on separate days. Gonococci wereiron stressed as previously reported (2) prior to experimentation.SDS-PAGE and Western blotting of whole-cell lysates were routinelyperformed to determine that cultures were consistently and equivalentlyiron stressed, as evidenced by reactivity with anti-FrpB monoclonalantibody and/or anti-Tbp1 antisera. Iron-uptake assays were performed aspreviously reported (9) with the following modifications. Filters wereblocked just prior to experimentation with 30 μl, 10 mg/ml BSA in 1XCDM.Assays were performed in 200 μl volumes in 96 well filtration plates(MAHV Millipore, Bedford, Mass.) at 35° C. in a 5% CO₂ atmosphere.Potassium cyanide was dissolved in 1XCDM. The vacuum manifold was fromMillipore Multiscreen Assay System. Heme was used at 0.5 μM, transferrinat 6.25 μM, and citrate at 100 μM. Membranes were air dried overnight,and the Millipore punch kit was used to separate and collect individualfilters prior to counting. Data were expressed as counts per minute perμg of protein.

[0088] Preparation of aerobactin and enterobactin. Purified aerobactinand enterobactin were the generous gift of P. E. Klebba. Aerobactin wasferrated as follows. Ferric sulfate was dissolved to 4 mM in 50 ml ddH₂Ocontaining 1.5 μl HCl. 400μ 4 mM aerobactin was added to 400 μl 4 mMferric sulfate and 80 μl 0.5M Na₂HPO₄. The ferri-aerobactin was run overa CM-cellulose (Sigma, St. Louis, Mo.) column equilibrated in 0.05MNa₂HPO₄. The final concentration of aerobactin was determined by readingthe absorbance at 400 nM (24).

[0089] Iron sources. Human transferrin, human lactoferrin, bovine heme,human hemoglobin, and human haptoglobin were obtained from SigmaChemical Co. (St. Louis, Mo.). ⁵⁵Fe hemin was purchased from the customsynthesizing facility at NEN Products Dupont (Wilmington, Del.) lotnumber FE55.1193RS. Transferrin, lactoferrin, and citrate were ferratedwith ⁵⁵FeC1 as previously described (36).

[0090] RNase assay. The RNase assay was performed as previouslydescribed (71), except 0.1 N HCl was used instead of 0.5N HCl.

[0091] Hemin affinity purification. Hemin agarose was purchased fromSigma Chemical Co. (St. Louis, Mo.). The method of affinity purificationwas described by Lee (33).

[0092] Bactericidal assays. Bactericidal assays were performed asdescribed previously (18).

[0093] Cloning the gonococcal frpB gene. Sarcosyl insoluble membranefractions from gonococcal strain UU1008 were used to obtain FrpBN-terminal amino acid sequence (see above). A degenerate oligonucleotidecontaining inosine (designated MB.3, shown in FIG. 1) was deduced fromthis sequence and used to probe a Southern blot of FA19 chromosomal DNA.Each restriction digest contained a single hybridizing band. A 5.8 kbDra I fragment was chosen for further analysis.

[0094] A λZapII library containing EcoRI-linkered FA19 chromosomal Dra Ifragments (2) was screened with oligo MB.3. Approximately one positiveplaque was identified for every 10,000 plaques screened. Attempts toexcise the phagemid containing the intact insert consistently resultedin deletion products smaller than pBluescript II SK⁺ alone. Since such alarge chromosomal fragment potentially contained both the frpB promoterand entire frpB coding sequence and that the expression of FrpB might betoxic in E. coli, smaller fragments were subcloned into pBluescript IISK⁺.

[0095] DNA prepared from one of the positively hybridized plaques,λfrpB-4(FIG. 2), was digested with EcoRI to release the insert DNA. Theexpected 5.8 kb fragment was isolated from an agarose gel and furtherdigested with Cla I to generate a 540 bp, MB.3-hybridizing fragment andan approximately 5.3 kb fragment which did not hybridize to MB.3. Thesmaller fragment ligated into pBluescript II SK⁺ was stable in E. coliDH5αMCR and was designated pUNCH319. The larger fragment ligated intopBluescript II SK⁺ generated pUNCH320. pUNCH320 caused E. coli DH5αMCRto grow poorly and appeared to be severely restricted in copy number.These data suggested that other sequences located 3′ of frpB may also betoxic to E. coli and that further subcloning was necessary to obtainstable clones. Digestion of pUNCH320 with Mlu I and EcoR I releasedfragments of approximately 1.0 kb and 1.5 kb, leaving a 2.8 kb Cla I-MluI fragment attached to pBluescript II SK⁺. This 5.8 kb fragment (vectorplus 2.8 kb Cla I-Mlu I insert) was subsequently isolated, treated withKlenow, and re-ligated to itself to generate pUNCH325. DH5αMCR(pUNCH325) transformants were stable and the plasmid copy numberapparently normal.

[0096] Nucleotide sequence and analysis of frpB. PCR amplification ofchromosomal DNA followed by sequence analysis of clones confirmed theCla I junction between pUNCH319 and pUNCH325. The combined nucleotidesequence and deduced amino acid sequence from pUNCH319 and pUNCH325 areshown in FIG. 3. Putative promoter sequences were located upstream of awell conserved Fur box (4). A string of nine cytosine residues was notedbetween the putative −10 and −35 RNA-polymerase binding sites. AShine-Dalgarno sequence starting at nucleotide 307 and ending atnucleotide 310 (FIG. 3), was located six bases before an ATG codon, thestart of a 1,925 bp open reading frame (ORF). This ORF encoded a proteinof 713 amino acids. The predicted protein contained a typical signalsequence and characteristic Ala-X-Ala, signal peptidase I cleavage site.The first ten amino acids adjacent to the cleavage site were identicalto the peptide sequence obtained from the mature FrpB protein. Aclassical TonB box was noted at residues 32-36. The mature protein had acalculated molecular weight of 76.6 kD and an isoeletric point of 10.38.The sequence downstream of the ORF revealed an inverted repeat but nostring of T residues characteristic of rho-independent transcriptiontermination (69). The protein terminated with an aromatic residuepreceded by nine alternating hydrophobic and hydrophilic amino acids.This structure is typical of many bacterial outer membrane proteinssequenced to date (58).

[0097] GenBank homologies. Comparison of FrpB with other sequences inGenBank revealed some interesting homologies. Several regions of thepredicted FrpB protein shared similarity with regions identified inother proteins as potentially important for membrane localization and/orTonB interaction. Localized homology was found between FrpB and thefamily of TonB-dependent outer membrane receptor proteins including BtuB(25) and FepA (35) of E. coli and between Tbp1 (13) and IroA (42) ofNeisseria species. This similarity was limited to the highly conserveddomains (13), and suggested that FrpB may also be a TonB-dependentreceptor. More similarity was found with HemR, the hemin receptor ofYersinia enterocolitica (55). HemR is an iron-regulated, outer membraneprotein that is also a member of the family of TonB-dependent receptorproteins. Overall the two proteins were 26% identical and 48% similar.The most notable similarity was seen with CopB, a major outer membraneprotein of Moraxella catarrhalis (26). Overall FrpB and CopB were 52%identical and 71% similar.

[0098] Transposon mutagenesis of frpB. In order to construct FrpBmutants, the gonococcal insert in pUNCH319 was ligated into pUP1(19),creating pUNCH321. The Ω fragment from pHP45 Ω was ligated into a uniqueBgl I site in pUNCH321 (Insertion site shown in FIG. 3). This DNA wasreintroduced into the chromosome of gonococcal strain FA19 bytransformation and allelic replacement, creating FA6807. Southern blotanalysis of chromosomal DNA from FA19 and FA6807 indicated that a 450bp, MB.3-hybridizing, HincII fragment present in the parent was missingin FA6807 and a new reactive band of approximately 2.5 kb was present(FIG. 4, panel A). An identical blot (FIG. 4, panel B) probed with Ω,only hybridized to the 2.5 kb fragment in FA6807. SDS-polyacrylamide gelelectrophoresis (SDS-PAGE) and Western blot analysis with anti-FrpBmonoclonal antibody W.6, confirmed that FrpB was absent from this strain(FIG. 5).

[0099] The Ω insertion in frpB was also introduced into FA6747(tbpA::mTn3(Cm)) by transformation and allelic replacement creatingFA6808. The FrpB⁻/Tbp1⁻ phenotype of FA6808 was confirmed by SDS-PAGEand Western blot analysis. This strain was used for FrpB functionanalysis as described below.

[0100] Utilization of iron sources. In an attempt to determine thefunction that FrpB plays in iron utilization, FA19 and FA6807 were grownin chemically-defined media (CDM) lacking iron. Aliquots ofiron-stressed cultures were plated onto CDM agarose containing 10 μMDesferal and GC base agar containing 50 μM Desferal. Sterile 3 mm discscontaining either citrate, transferrin, lactoferrin, heme, hemoglobin,or hemoglobin bound to haptoglobin were positioned around each plate.One disc without any added iron source was added as a negative control.After overnight incubation, growth of both strains was evident aroundall discs except the negative control.

[0101]N. gonorrhoeae can utilize aerobactin (67) and enterobactin (45)as iron sources. To determine if FrpB functioned as either an aerobactinor enterobactin receptor, FA19, FA6808, FA6747, KDF541, KDF541/pABN6,and BN1071 (Table 1) were iron stressed in CDM as above and plated ontoCDM agarose containing 2.5 μM 30% iron-saturated transferrin. FA6747 andFA6808 could not use Tf as an iron source because they lacked Tbp1,therefore these strains could grow only in the presence of a functionalhigh-affinity siderophore receptor. Three sterile discs were positionedaround each plate. Either 30% saturated lactoferrin (positive controlfor gonococcal viability) or filter-sterilized, iron-free supernatantfrom LG1315 pColV (aerobactin producer) or AN102 (enterobactinhyper-producer) were added to each disk. After overnight incubation, E.coli controls grew as expected suggesting that both siderophores wereefficient at stripping iron from transferrin, the sole iron sourceprovided in the media. FA19 grew over the entire transferrin plate asexpected, however, growth of FA6808 and FA6747 was only evident aroundthe lactoferrin disks, suggesting that the cells were viable but unableto use aerobactin or enterobactin under these conditions.

[0102] Aerobactin utilization by FA19 and FA6807 was further evaluatedin chemically-defined, liquid media, employing various concentrations ofpurified ferri-aerobactin (FIG. 6). The aerobactin receptor-negative E.coli strain KDF541 and aerobactin receptor-positive E. coli strainKDF541 (pABN6) were used as controls. These data suggested that N.gonorrhoeae FA19 and FA6807 used ferri-aerobactin similarly and in aconcentration-dependent fashion analogous to the aerobactinreceptor-negative E. coli control. Growth stimulation of gonococci byferri-aerobactin required relatively high concentrations (3 μM) andnever attained a density equivalent to that of the Tf or citratecontrols. These experiments confirmed the ability of gonococci toutilize ferri-aerobactin as an iron source in vitro but showed that thisability was not dependent upon a high-affinity receptor-mediated event.

[0103]⁵⁵Fe uptake from hemin, Tf, and citrate. Because of the highdegree of similarity between HemR, a known hemin receptor in Y.enterocolitica and FrpB, it was analyzed whether a quantitativedifference in ⁵⁵Fe uptake from hemin could be detected between FA19 andFA6807. Uptake of ⁵⁵Fe from transferrin by FA19, FA6807, and the Tbp1mutant FA6747 were used as controls. The results indicated that while⁵⁵Fe uptake from transferrin was approximately wild type inFA6807(P=0.826), ⁵⁵Fe uptake from hemin was reduced by approximately 60%(P<0.001)(FIG. 7). Surprisingly, ⁵⁵Fe uptake from hemin was alsosignificantly reduced in FA6747 (P<0.001). To determine whether theinability to use ⁵⁵Fe from hemin was specific to FA6807(FrpB⁻) andFA6747 (Tbp1⁻), ⁵⁵Fe uptake from hemin was assayed in otherwell-characterized, gonococcal mutants specifically altered in theexpression of other iron-repressible proteins. The Tbp2⁻ and Lbp⁻strains, FA6819 and FA6775 respectively, were also reduced in ⁵⁵Feinternalization from hemin (P<0.001). These data suggested that eithermore than one protein was involved in the internalization of hemin ironor the notable decrease in hemin-iron uptake in these mutants resultedfrom unanticipated, non-specific effects of each of these mutations on aseparate membrane-bound, heme-iron-uptake system.

[0104] Reconstruction of frpB in pACYC184 and functional complementationof RK1065(hemA). In an attempt to determine if FrpB could function as aheme receptor, an E. coli hemA mutant was complemented with FrpB.Although expression of FrpB from the high copy-number vector pBluescriptII SK⁺ was toxic to E. coli, expression from the low copy-number vectorpACYC184 was tolerated. The frpB reconstruction strategy is outlined inFIG. 8. Briefly, the insert from pUNCH319 was ligated into the Cla I andBamH I sites of pACYC184, generating pUNCH330. pUNCH330 was digestedwith Cla I and the gel-purified Cla I-Xba I fragment from pUNCH325 wasligated into this site as follows. After ligating for four hours, Klenowwas added to the ligation mixture for 30 minutes at room temperature torepair non-ligated Cla I and Xba I ends. The reaction was furtherligated overnight. The frpB clone in pACYC184 was designated pUNCH331.FrpB expression from pUNCH331 was iron repressible, suggestingregulation by E. coli Fur.

[0105] RK1065 is an E. coli hemA mutant which is unable to synthesize orinternalize heme (27). Growth stimulation requires eitherδ-aminolevulinic acid, or heme and a functional heme receptor.Transformation of pUNCH331 into RK1065 supported growth on heme plates,whereas pACYC184 alone did not (FIG. 9). An Rnase leakage assay wasperformed to determine if FrpB expression altered the E. coli outermembrane, thereby allowing heme to simply diffuse into the cell (71).The E. coli strains C386 and HB101 containing pEBH21 were used aspositive and negative controls respectively. No difference in leakinesswas detected between RK1065 (pACYC184) and RK1065 (pUNCH331), suggestingthat growth of RK1065 (pUNCH331) on heme plates was not due to amembrane perturbation gross enough to permit leakage of the periplasmicprotein RNase H. Nevertheless, RK1065 (pUNCH331) was more sensitive toseveral hydrophobic antibiotics than the same strain with pACYC194 alone(FIG. 9). This experiment suggested that the presence of FrpB in E. coliprobably allowed heme to enter non-specifically either by creating apore or by perturbing the integrity of the outer membrane. Uptake of⁵⁵Fe from hemin in RK1065 (pUNCH331) was not inhibited by KCN,consistent with a non-specific, non-receptor mediated mechanism ofuptake.

[0106] Bactericidal Assay. In M. catarrhalis, CopB, the protein with thegreatest similarity to FrpB, appears to play a major role in serumresistance. Mutants which are missing CopB have decreased serumresistance. Mutants which are missing CopB have decreased serumresistance and survival in a mouse model (26). Standard bactericidalassays were performed with normal human serum on FA19 and FA6807 grownunder iron-limiting conditions and were unable to detect any differencein survival; both strains were completely serum resistant. TABLE 1Bacterial strains, plasmids and phage. Strain, plasmid or phageDescription Source/reference FA19 Wild type [Mickelsen, 1981 #38] FA6807frpB: :Ω(FrpB^(.)) This study FA6808 frpB: :Ω tbpA: :mTn3(Cm) (FrpB^(.),Tbp1^(.)) This study FA6747 tbpA: :mTn3(Cm) (Tbp1^(.)) [Cornelissen,1992 #13] FA6819 ΔtbpB (Tbp2^(.)) [Anderson, 1994 #2] FA6775 lbpA::mTn3(Cm) (Lbp^(.)) [Biswas, 1994 #6] UU1008 Wild type Zell McGeeDH5αMCR F^(.) mcrA mcrB mrr φ80dlacZΔM15 Δ(argF-lac)U169 BethesdaResearch Labs recAl endAl hsdR hsdM supE44 λ^(.)thi-1 gyrA96 relAIBN1071 F^(.), pro, trp, rslL, entA (Ent^(.), FepA⁺) [Klebba, 1982 #30]AN102 BN1071, leu, fepA (Ent⁺, FepA^(.)) [Klebba, 1982 #30] KDF541BN1071, entA, fepA (Ent^(.), FepA^(.)) [Rutz, 1992 #46] KDF541/pABN6(Ent^(.), FepA^(.),luc^(.)) [de Lorenzo, 1987] LG1315/pcolV BN1071, cir(lutA⁺, luc+) [Warner, 1981 #63] RK1065 hemA R. Kadner HB101 F^(.),hsd20 (r_(B) ^(.),m_(B) ^(.)), recA13, ara-14, proA2, lacYl, galK2,Maniatis et. al. 1982 rpsL20 (Sm^(r)), xyl-5, intl-1, supE44, λ^(.) C386ompA lpp [Sonntag, 1978 #53] pACYC184 ori p15a, Cm^(R, Tc) ^(R) NewEngland Biolabs pBluescript II SK+ on pMB1, AP^(R) Stragene pHP45Ωsource for the Ω fragment (Sm^(R)) [Prentki, 1984 #44] pUP1 pHSS6containing gonococcal uptake seqeunce (Kan^(R)) [Elkins, 1991 #19]pEBH21 pBC II SK⁺ derivative (Cm^(R)) [Hardham, 1994 #22] pUNCH319pBluescript II SK⁺containing 540 bp EcoR I-Cla I fragment This Studyfrom λfrpB.4 pUNCH320 pBluescript II SK⁺containing 5.3 kb Cla I-EcoRIfragment This Study from λfrpB.4 pUNCH321 pUP1 containing 540 bp EcoRI-Cla fragment from This Study pUNCH319 pUNCH324 pUNCH321 containing Ωfragment from pHP45Ω in unique This Study Bgl I site pUNCH325pBluescript II SK⁺containing 2.8 kb Cla I-Mlu I fragment This Study frompUNCH320 pUNCH330 540bp EcoR I-Cla fragment from pUNCH319 in This StudypACYC184 pUNCH331 reassembled gonococcal frpB gene in pACYC184 ThisStudy λZapII excisable lambda phage vector Statagene

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What is claimed is:
 1. An isolated nucleic acid molecule that encodes anamino acid sequence comprising a FrpB protein.
 2. An isolated nucleicacid molecule of claim 1 comprising the nucleotide sequence of FIG. 3.3. An isolated nucleic acid molecule of claim 1 comprising thenucleotide sequence of FIG.
 10. 4. An isolated nucleic acid molecule ofclaim 1, wherein the FrpB protein is the FrpB protein of Neisseriagonorrhoeae.
 5. An isolated nucleic acid molecule of claim 1, whereinthe FrpB protein is the FrpB protein of Neisseria meningitides.
 6. Apolypeptide encoded by the isolated nucleic acid molecule of claim
 2. 7.A polypeptide encoded by the isolated nucleic acid molecule of claim 3.8. A vector which comprises the nucleic acid molecule of claim
 1. 9. Avector of claim 8, wherein the nucleic acid molecule is linked to aplasmid.
 10. A host vector system for the production of a polypeptidehaving the biological activity of a FrpB antigenic polypeptide whichcomprises the vector of claim 8 in a suitable host.
 11. A host vectorsystem of claim 10, wherein the suitable host is a bacterial cell oranimal cell.
 12. A method of producing a polypeptide having thebiological activity of a FrpB antigenic polypeptide which comprisesgrowing the host vector system of claim 10 under suitable conditionspermitting production of the polypeptide and recovering the polypeptideso produced.
 13. A method of producing a vaccine composition thatprotects a mammal from infection by N. gonorrhoeae comprising combiningthe FrpB protein encoded by the isolated nucleic acid of claim 1 with apharmaceutically acceptable carrier.
 14. The method of claim 13 furthercomprising combining the FrpB with an effective amount of an adjuvant.15. The method of claim 13, wherein the amino acid sequence of thepolypeptide comprises the FrpB protein of N. gonorrhoeae.
 16. The methodof claim 13, wherein the mammal is a human.
 17. A method of producing avaccine composition that protects a mammal from infection by N.meningitidis comprising combining the FrpB protein encoded by theisolated nucleic acid of claim 1 with a pharmaceutically acceptablecarrier.
 18. The method of claim 17 further comprising combining theFrpB with an effective amount of an adjuvant.
 19. The method of claim17, wherein the amino acid sequence of the polypeptide comprises atleast a portion of the FrpB protein of N. meningitidis.
 20. The methodof claim 17, wherein the mammal is a human.
 21. A vaccine compositioncapable of protecting a mammal against infection by N. gonorrhoeae, thevaccine composition comprising the FrpB protein encoded by the isolatednucleic acid of claim 1 and a pharmaceutically acceptable carrier. 22.The vaccine composition of claim 21 further comprising an effectiveamount of an adjuvant.
 23. The vaccine composition of claim 21, whereinthe amino acid sequence of the polypeptide comprises at least a portionof the FrpB protein of N. gonorrhoeae.
 24. The vaccine composition ofclaim 21, wherein the mammal is a human.
 25. A vaccine compositioncapable of protecting a mammal against infection by N. meningitidis, thevaccine composition comprising the FrpB protein encoded by the isolatednucleic acid of claim 1 and a pharmaceutically acceptable carrier. 26.The vaccine composition of claim 25 further comprising an effectiveamount of an adjuvant.
 27. The vaccine composition of claim 25, whereinthe amino acid sequence of the polypeptide comprises at least a portionof the FrpB protein of N. meningitidis.
 28. The vaccine composition ofclaim 25, wherein the mammal is a human.
 29. A method of protecting amammal against infection by N. gonorrhoeae comprising administering tothe mammal a vaccine composition of claim
 21. 30. A method of protectinga mammal against infection by N. meningitidis comprising administeringto the mammal a vaccine composition of claim
 25. 31. An antibodydirected to an epitope of the FrpB protein encoded by the isolatednucleic acid sequence of claim
 2. 32. An antibody directed to an epitopeof the FrpB protein encoded by the isolated nucleic acid sequence ofclaim
 3. 33. A method of detecting an antibody specific for N.gonorrhoeae in a sample comprising: (a) contacting the sample with aFrpB protein encoded by the isolated nucleic acid sequence of claim 4under conditions to form a complex between the polypeptide and theantibody; and (b) detecting any complex so formed; thereby detecting anantibody specific for N. gonorrhoeae.
 34. A method of claim 33, whereinthe FrpB protein is labeled with a detectable marker.
 35. A method ofdetecting an antibody specific for N. meningitidis in a samplecomprising: (a) contacting the sample with a FrpB protein encoded by theisolated nucleic acid sequence of claim 5 under conditions to form acomplex between the polypeptide and the antibody; and (b) detecting anycomplex so formed; thereby detecting any antibody specific for N.meningitidis.
 36. A method of claim 35, wherein the FrpB protein islabeled with a detectable marker.
 37. A method of treating a mammalinfected by N. gonorrhoeae comprising administering to the mammal anantibody of claim
 31. 38. A method of treating a mammal infected by N.gonorrhoeae comprising administering to the mammal an antibody of claim32.
 39. The method of claim 37 or 38 wherein the mammal is a human. 40.The method of claim 37 or 38 wherein the antibody is monoclonal.
 41. Amethod of treating a mammal infected by N. meningitidis comprisingadministering to the mammal an antibody of claim
 31. 42. A method oftreating a mammal infected by N. meningitidis comprising administeringto the mammal an antibody of claim
 32. 43. The method of claim 41 or 42wherein the mammal is a human.
 44. The method of claim 41 or 42 whereinthe antibody is monoclonal.